Macromolecular neuraminidase-binding compounds

ABSTRACT

The invention provides novel macromolecules, methods for their preparation, pharmaceutical formulations thereof and their use as anti-influenza agents. The invention also provides a novel diagnostic method which can be used for detection of all types of influenza A and B virus. The macromolecular compound of the invention has attached to it one or more molecules (neuraminidase binders) which bind to the active site of influenza virus neuraminidase but which are not cleaved by the neuraminidase.

This invention relates to a new class of chemical compounds and theiruse in medicine. In particular the invention provides novelmacromolecules, methods for their preparation, pharmaceuticalformulations thereof and their use as anti-influenza agents. Theinvention also provides a novel diagnostic method which can be used fordetection of all types of influenza A and B virus.

BACKGROUND OF THE INVENTION

Influenza A and B viruses are major causes of acute respiratory disease,resulting in an estimated 30-50 million infections annually in theUnited States alone. Influenza A has been responsible for majorepidemics, such as the “Spanish Flu” of 1919 which killed millions ofpeople. Influenza remains a difficult disease to control, resulting insignificant morbidity, and mortality largely due to secondary infectionin eldery or debilitated patients. Vaccines are continually beingrendered obsolete by antigenic shift or drift, and consequentlyimmunization is only about 70% effective in preventing infection. Theonly drugs approved by regulatory authorities for treatment of influenzaare amantidine and rimantidine, which are ineffective against influenzaB, and are known to have serious side-effects.

Many viral and bacterial infections may present with symptoms similar tothose of influenza. The rapid identification of respiratory viruseswould enable physicians to use the most appropriate therapy early in theillness. For example, an early and accurate diagnosis would allowdecisions regarding the use of antibacterial therapy and hospitalisationof children and the elderly.

Laboratory tests for the identification of viruses in clinical materialare widely used, and a variety of different detection methodology isavailable. The textbook, “Laboratory Diagnosis of Viral Infections”,Marcel Dekker 1992, Ed E. H. Lennette generally discusses methods whichare used for a wide range of viruses, including influenza virus.

A number of tests are available for the diagnosis of influenza A and B.The traditional method of identifying influenza viruses has been the useof cell culture, which is highly sensitive and specific. Unfortunately,the time required for culture, isolation and identification of influenzavirus can range between 2 and 10 days, thus making it virtually uselessin guiding the physician to an appropriate therapy. Since influenzavirus infection is normally self-limited, diagnosis must be rapid iftherapy is to be effective.

In addition to the cell culture methods for detecting influenza, therehave recently become available a few rapid direct tests, which arespecific for influenza A. Thus, a monoclonal immunofluorescence assay(IFA) has been reported (Spada, B. et al, J. Virol. Method, 1991 33 305)and at least one rapid enzyme immunoassay (EIA) is available(Ryan-Poirier, K. A. et al, J. Clin. Microbiol., 1992 30 1072). A numberof comparisons of these rapid detection methods for influenza A havebeen reported; see for example Leonardi, G. P. et al, J. Clin.Microbiol., 1994 32 70, who recommended that direct specimen testing beused together with culture isolation, so as to permit bothidentification of the virus in time to institute therapy and infectioncontrol measures, and to monitor the antigenic constitution of influenzastrains prevalent in the community.

The IFA method is reported to be labor-intensive, and requiresconsiderable technical expertise, with the results often being difficultto interpret. On the other hand, the EIA (Directigen FLU-A; BectonDickinson Microbiology Systems) method gave a high level offalse-positive results, and it has been recommended that this assayshould be used in laboratories only as an addition or substitute fordirect immunofluorescence tests (Waner, J. L. et al, J. Clin.Microbiol., 1991 29 479).

As well as the problems mentioned above with the currently availablerapid assays for influenza, there are other fundamental deficiencies insome of these methods. Firstly, none of the available assays can detectinfluenza B, which means that even a negative test result would leavethe physician uncertain about the type of therapy that should be used.Secondly, if a rapid immunoassay method depends on the use of antibodiesto one of the influenza A proteins, there may be a serious problem indetecting new strains of the virus which have undergone a drift or shiftin the structure of the antigenic proteins. Influenza A is notorious forits propensity to undergo such changes.

Another type of rapid assay for influenza viruses has been described ina series of patent specifications (see for example Liav, A. et al, PCTPatent Application No. 92/12256). The method involves the use of achromogenic substrate for the influenza neuraminidase enzyme. In otherwords the assay depends on visualising a dye, which is formed when theinfluenza neuraminidase cleaves a special sialic acid-dye conjugatemolecule. This technique appears to offer limited specificity, becauseit could not readily distinguish between the presence of viralneuraminidase and other forms of the enzyme, particularly bacterialneuraminidase. It may also have low sensitivity because of therelatively slow activity of viral neuraminidase.

Influenza A and B have two major surface glycoproteins, hemagglutinin(HA) and the enzyme neuraminidase (NA), which are both essential forinfectivity. It is believed that HA is necessary for the virus to attachto cells whereas NA is needed for release of the virus from cellsurfaces. There are typically about 600 trimeric HA and about 50 copiesof the NA tetramer units on the surface of each virus particle. Both HAand NA therefore are attractive potential targets in the search foranti-influenza drugs, but to date no anti-influenza drugs that work ateither of these sites are available for clinical use.

Influenza virus hemagglutinin binds to the sialic acid containingglycoproteins and glycolipids on cell-surface receptors, therebyinitiating the process of attachment of the virus to a cell andsubsequent infection. The strength of the binding of a virus particle tothe cell membrane appears to depend on the interaction of multiplecopies of the influenza HA with multiple sialic acid groups on the cellsurface.

Using this concept of a polyvalent interaction, several workers havereported the synthesis of macromolecules containing two or more sialicacid derivatives which act as hemagglutinin inhibitors. Although somestrong HA inhibitors have been discovered, none of these polyvalentmacromolecules has been shown to prevent influenza infection in vivo.Recent papers by Whitesides and co-workers (J. Amer. Chem. Soc., 1996118 3789-3800; J. Medicinal Chem., 1995 38 4179-4190) have summarisedthe various efforts which have used this approach to the design ofinhibitors of influenza hemagglutinin.

There are several known inhibitors of NA, most of which are closeanalogues of neuraminic acid, the enzyme's natural substrate, such as2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) (Meindl et al,Virology, 1974 58 457-63). International Patent Application No. WO91/16320 describes analogs of DANA which are very active, both in vitroand in vivo, against influenza A and B neuraminidase. One of thesecompounds (Compound I, designated GG167 or 4-guanidino-Neu5Ac2en) is inclinical trial, and shows promise for the treatment of influenza(Hayden, F. G. et al, J. Amer. Med. Assoc., 1996 275 295).

More recently, aromatic compounds with neuraminidase-inhibitory activityhave been described in U.S. Pat. No. 5,453,533 by Luo et al and U.S.Pat. No. 5,512,596 by Gilead Sciences, Inc., and analogues of compound(I), in particular compounds in which the side-chain at carbon 6 isether-linked, have been described in International Patent ApplicationNo. WO 96/26933 by Gilead Sciences, Inc. and in C. Kim et al, J. Amer.Chem. Soc., 1997 119 681.

Several research groups have attempted to find simpler or more potentanalogues of compound (I), but reports to date (e.g. Bamford M. J., J.Chem. Soc. Perkin Trans. I, 1995, 1181) indicate that any changes to thestructure of compound (I), particularly at the glycerol side chain, arelikely to reduce the neuraminidase-binding properties. In addition, incontrast to the situation with HA, there do not appear to be any knownmacromolecular or polymeric inhibitors of neuraminidase. Sialicacid-containing polymers have been described in U.S. Pat. No. 5,192,661by Roy et al and in U.S. Pat. No. 5,571,836 by Bovin et al, but thesecompounds were synthetic polysialosides designed for use as antigens oras binders of haemagglutinin.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides macromolecular compounds whichhave attached to them one or more molecules which bind to the activesite of influenza virus neuraminidase; these molecules are referred toherein as “neuraminidase binders”. Preferably the neuraminidase binderis attached to the molecule via a spacer or linker group so that theneuraminidase binder is not sterically hindered by the backbone of themacromolecule. The neuraminidase binder may be any agent which binds tothe active site of influenza virus neuraminidase, provided that it isnot cleaved by the enzyme. The binding need not be irreversible, but thebinding group should have a high binding affinity, preferably an IC₅₀ of10⁻⁶ M or less.

The invention particularly relates to a new class of chemical compoundsand their use as therapeutic and diagnostic agents for the treatment anddetection of influenza A and B. More specifically the invention concernsmacromolecules which have attached to them neuraminic acid (sialic acid)derivatives which bind to neuraminidase of influenza A or B, and whichoptionally also have a functionality which allows the compounds to bebound to a surface, or which can be used as a detectable label.

Surprisingly, we have found that when compound (I) is functionalisedthrough the 7-position of the sialic acid structure, it can be attachedto large synthetic or natural polymers to give complexes which inhibitinfluenza A and B neuraminidase, and which can prevent or inhibitinfluenza infection. Rather than destroying the influenzaneuraminidase-binding properties of compound (I), we find that whenmultiple numbers of this and similar compounds are linked through their7-position by a suitable spacer to a variety of macromolecules theaverage binding per sialic acid group is not substantially reduced. Thusthrough the binding to neuraminidase the macromolecules are tightlybound to the virus, and possibly because of the size and steric effectsof the complexes, the infectivity of the influenza virions is reduced.Such macromolecular compounds can also be used to enable the detectionof influenza A and B virus through their ability both to bind theinfluenza virus selectively and at the same time to be bound to asurface or to a detectable linking group.

The biological activity of the macromolecular compounds of the inventionand the diagnostic method of the invention are both based on the use ofligands on the macromolecules that are able to bind specifically to theactive site of influenza virus neuraminidase, or functionalisedderivatives of such compounds, as binding and/or detecting agents toidentify influenza virus in clinical specimens. The term “neuraminidasebinders” is used hereinafter to refer to these compounds and theirfunctionalized derivatives. The method and compounds of the inventioncan function either in the presence or the absence of compounds bindingnon-specifically to influenza virus neuraminidase.

In a preferred embodiment, the present invention provides a compound offormula (II):

(X—Y)_(n)—M—(Z)_(m)  (II)

wherein X is a neuraminidase-binding 2,3-dehydro-sialic acid derivative(2) which is linked at the 7-position via a spacer group Y to amacromolecule M, and Z is an optional extra substituent on themacromolecule.

The neuraminidase-binding moiety X is a sialic acid derivative offormula (2)

in which the spacer Y connects to the W group, and

wherein R represents an azido group, an unsubstituted or substitutedguanidino group, or an unsubstituted or substituted amino group;

R² represents COCH₃, COCF₃, SO₂CH₃ or SO₂CF₃;

W represents O(C═O)NH, O(C═S)NH, NH(C═O)NH or NH(C═S)NH and is attachedthrough the NH to group Y;

m is an integer between 0 and 1000; and

n is an integer between 1 and 1,000.

The spacer group Y is an optionally substituted chain of up to 1000atoms chosen from carbon, nitrogen, oxygen and sulphur.

The macromolecule M is a synthetic or natural polymer, protein, antibodyor enzyme of molecular weight from 10⁴ up to 10⁷.

The Y group is generally linked covalently to the macromolecule M, butmay also be bound through non-covalent attachment, for example when M isavidin and Y has a terminal biotin group.

The second and optional substituent Z can be a group that bindshemagglutinin, such as a 2-linked sialic acid derivative, or a groupthat can act as a detectable label, such as a biotin or fluorescentmolecule, or it can be an antibody-binding hapten. The optionalsubstituent z can also be an enzyme such as horseradish peroxidase (HRP)or alkaline phosphatase (AP) which can be used to enable detection ofinfluenza. Alternatively, the group Z can be a group with a terminalfunctionality that is suitable for binding the macromolecule to asurface, such as NH₂, SH, CO₂H, CHO, or CH═CH₂.

In another preferred embodiment, the invention provides neuraminidasebinders of formula (IIA):

(X′—Y)_(n)—M—(Z)_(m)  (IIA)

wherein X′ is a neuraminidase-binding cyclohexenyl derivative of formula(2a),

which is linked through the ether side-arm,

R, R²,Y, n and m are as defined above for formula (2), and

R¹ and W′ are lipophilic C₁-C₁₂ alkyl or alkylene groups which areoptionally substituted by one or more halogen atoms or alkoxy,haloalkoxy or optionally substituted aryl groups.

Suitable spacer groups Y include, but are not limited to, aminoalkylgroups, (poly)amino acids, linear peptides, oligosaccharides andpolysaccharides, polyethylene glycol units, and amino-dialkylureas, anyof which may be used alone or in combination. Typically the spacer groupY has a terminal amino group, which is used to form an amide or Schiffsbase linkage on to the macromolecule M.

Suitable substituents of the guanidino or amino groups R include methyl,ethyl, allyl, amino, cyano or nitro.

Suitable macromolecules M include proteins, enzymes, antibodies,water-soluble synthetic polymers such as polyacrylic acids andpolyacrylamides, polysaccharides and polyaminoacids. Macromoleculeswhich are particularly suitable for use in diagnostic applicationsinclude bovine serum albumin (BSA), horseradish peroxidase (HRP), avidinand related proteins such as streptavidin or neutravidin, andimmunoglobulins.

Macromolecules which are particularly suitable for use in compounds ofthe invention to be used in the treatment of influenza includepolysaccharides, synthetic polymers such as polyacrylamides,polyethylene glycols, polyureas, polyacids, polyesters, polyamides andvarious co-polymers such as N-(2-hydroxy-propyl)methacrylamide (HMPA),which are known to be safe for administration to humans. The personskilled in the art will be aware of other pharmaceutically-acceptablepolymers.

One preferred group of compounds of the invention comprises compounds(II), in which X is a GG167 derivative of formula (2) wherein:

R is guanidine, R² is acetyl, W is the group O(═CO)NH and the spacer Yis a chain made up of between 6 and 60 carbon, nitrogen and oxygenatoms.

The macromolecules of formula (II) are inhibitors of influenza A and Bneuraminidase and possess anti-influenza activity. Thus in a secondaspect of the invention there is provided a pharmaceutical compositionfor treatment of influenza A or influenza B comprising a compound of theinvention, preferably a compound of formula (II) or formula (IIA), or apharmaceutically-acceptable derivative thereof, together with apharmaceutically-acceptable carrier.

In a third aspect there is provided a method of treatment of influenzainfection in a mammal, including man, comprising the step ofadministering an effective amount of a compound of the invention,preferably a compound of formula (II) or formulae (IIA) or apharmaceutically acceptable derivative thereof, to a mammal in need ofsuch treatment.

There is also provided in a fourth aspect the use of a compound of theinvention, preferably a compound of formula (II) or formula (IIA) forthe manufacture of a medicament for the treatment of an influenza viralinfection.

The compounds of the invention may also be used in combination withother therapeutic agents, for example other anti-infective agents,particularly other anti-viral agents. The invention thus provides in afurther aspect a pharmaceutical composition comprising a compound of theinvention, preferably a compound of formula (II) or formula (IIA) or apharmaceutically acceptable salt or derivative thereof together with oneor more further therapeutically active agent, in particular ananti-viral agent, together with a pharmaceutically-acceptable carrier.

The invention also provides in another aspect a method of detection ofinfluenza virus, comprising the step of exposing a sample suspected toinclude said virus to a compound of the invention which is able to bindspecifically to the active site of influenza virus neuraminidase.

The method of the invention is applicable to all types of influenza Aand influenza B.

For the detection of influenza the compounds of the invention (II) maybe attached to a surface, either by covalent bonding or by non-specificbinding. The spacer group Y should be sufficiently long that theneuraminidase binding units X are exposed on the surface of theimacromolecule M and accessible to a virus particle.

For the detection of influenza the method of the invention may useselective capture with a compound of formula (II) and therebyconcentration of the virus, followed by detection of the virus using anyconvenient conventional method; the detection method need not haveinherent selectivity. For example, the binder (II) may be attached to asupport material, such as a membrane or polymer, such that virusparticles will be selectively captured and concentrated when a sample ispassed over or through the support. Therefore in one preferredembodiment of the invention, the group Z terminates in a functionalityable to bind to a surface. Many suitable functionalities are known inthe art.

Alternatively, a selective detection approach may be used; the virusparticles in a sample may for example be non-specifically captured andthen exposed to a macromolecular neuraminidase binder (II) whichincludes a detectable label Z, under conditions such that the binderattaches selectively to the influenza neuraminidase on the surface ofthe viral particle. The detectable label is then detected using anyconvenient method. For some detection systems, it is convenient to focusthe sample into a confined area, for example a spot or a line on asurface. This may be achieved by a variety of methods; for example, thesample may be suspended or non-selectively captured on to a filter orother support material, and then exposed to the labelled binder asabove.

In another alternative aspect the invention may use a combination ofselective capture and selective detection to provide a simple andsensitive two-stage method of detecting influenza virus. This makes useof the fact that influenza virus particles typically have about onehundred neuraminidase molecules spread over their spherical surface(White, D. O., Curr. Top. Microbiol. Immunol., 1974 63 1-48), and cantherefore attach to more than one binder at the same time.

Thus, a neuraminidase binder compound (II) may be attached to a support,for example as a narrow band across a length of porous membrane. Thetest sample is then applied at the other end of the membrane and allowedto flow across the band of bound compound. Any influenza virus particlesin the test sample will be trapped by the membrane-bound compound (II)and thus retained in the narrow band. In the second stage of the test, adetectable label attached to another neuraminidase binder (II) isallowed to flow through the membrane across the band of bound influenzavirus particles. The presence of influenza virus is then shown by anobservable change in the membrane at the site of the bound compound. Itis contemplated that the method and compounds of the invention aresuitable for use with the Biostar Optical Immunoassay (OAI) platform,which is described inter alia in U.S. Pat. No. 5,418,135 by Miller etal.

A very large number of suitable detection systems is known in the art,for example biotin-streptavidin, enzymic systems such as horseradishperoxidase or alkaline phosphatase, fluorescence systems,chemiluminescence systems, colloidal gold, radioactive labels andagglutination systems. It is contemplated that colloidal gold coatedwith a compound of the invention (II) will be a particularly convenientdetectable label. Similarly, compounds of the invention wherein themacromolecule M is horseradish peroxidase are expected to be ideal forthe ready detection of influenza. The skilled person will readily beable to select a suitable detection system and to optimise conditionsfor detection, using normal trial and error experimentation.

The compounds of the invention of formula (II) and theirpharmaceutically acceptable salts and derivatives may be prepared byvarious methods which include those described below. The methods ofpreparation outlined below form another aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

Examples of compounds of the invention include those of Formula (3) inwhich M is a protein, as listed in Table 1 below.

TABLE 1

Compound No. Spacer Y Protein M n Substituent Z No of m 3a(CH₂)₆NHCO(CH₂)₅NHCO(CH₂)₆CO— BSA 30 Biotinamidocaproyl  8 3b(CH₂)₆NHCO(CH₂)₅NHCO(CH₂)₆CO— Bovine IgG 60 Biotinamidocaproyl 18 3c(CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅NHCO(CH₂)₆CO BSA 6 — — 3d(CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅NHCO(CH₂)₆CO BSA 12 — — 3e(CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅NHCO(CH₂)₆CO HRP 3 — — 3f(CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅NH— HRP 2 — — 3g(CH₂)₆NH[CO(CH₂)₅NH]₄CO(CH₂)₆CO Bovine IgG 42 — — 3h(CH₂)₆NH[CO(CH₂)₅NH]₄CO(CH₂)₆CO Bovine IgG 12 — — 3i(CH₂)₆NH[CO(CH₂)₅NH]₄CO(CH₂)₆CO Bovine IgG 24 — — 3j(CH₂)₆NHCO₂[CH₂CH₂O]₇₀CH₂CH₂NHCO(CH₂)₆CO Bovine IgG 20 — —

Further examples of compounds of the invention include those of Formula(4) in which M is the protein avidin, which non-covalently binds thegroup X—Y, and also has covalently attached ligands Z as listed in Table2 below.

TABLE 2

Compound No. Spacer Y n Substituent Z m 4a(CH₂)₆NH(COCH₂NH)₂COCH₂NH-Biotin- 4 — — 4b(CH₂)₆NH(COCH₂NH)₂COCH₂NH-Biotin- 4 Biotinamidocaproyl  5 4c(CH₂)₆NH(COCH₂NH)₂COCH₂NH-Biotin- 4 Biotinamidocaproyl 10 4d(CH₂)₆NH(CO(CH₂)₅NH)₃CO(CH₂)₅NH-Biotin 4 Biotinamidocaproyl 10 4e(CH₂)₆NHCO(CH₂)₅NHCOCH₂(OCH₂CH₂)₁₆NH-Biotin 4 Biotinamidocaproyl  8

Further examples of compounds of the invention in which M is a syntheticpolymer include those of Formula (5) as shown below in Table 3, whereinthe substituents on the sialic acid group (2) are R²=Ac, R=guanidine andW is OCONH.

TABLE 3

Compound No. Spacer Y n p Substituent. Z m 5a (CH₂)₆ 1 13 — — 5b (CH₂)₆1 7 — — 5c (CH₂)₆NHCONH(CH₂)₆ 1 9 — — 5d (CH₂)₆NH(CO(CH₂)₅NH)₃CO(CH₂)₅ 18 — — 5e CH₂CH₂OCH₂CH₂OCH₂CH₂ 1 8 — — 5f (CH₂)₆ 1 500 — — 5g(CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅ 1 20 — — 5h (CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅1 50 — — 5i (CH₂)₆NH[CO(CH₂)₅NH]₃CO(CH₂)₅ 1 45 benzyl 5 5j (CH₂)₆ 1 50 —— 5k (CH₂)₆ 1 45 benzyl 5 5l (CH₂)₆ 2 40 hexyl-biotin 1 5m (CH₂)₆ 2 40hexyl-fluorescein 1 5n (CH₂)₆ 1 20 CH₂CH₂SH 1

Further examples of compounds of the invention in which M is a dextranbackbone (Molecular weight 500,000) include those represented by formula(6) as shown below in Table 4, wherein the substituents on theneuraminidase binding group (2) are R²=Ac, R=guanidine and W is OCONH.The integers n, m and p in the formula (6) give the percentage of theglucose units in the dextran backbone which are substituted by theparticular groups, ie when n, m and p do not add up to 100 the remainingdextran backbone is made up of unsubstituted glucose units. It shouldalso be remembered that there are three possible points of attachmentfor the various ligands bound to each unit of the dextran backbone.

TABLE 4

Compound No. Spacer Y n % Link V Substituent Z m % V′ p % 6a (CH₂)₆ 4C═O benzyl 17 H — 6b (CH₂)₆ 3 CH₂CO benzyl 17 CH₂CO₂H 20 6c(CH₂CH₂O)₂CH₂CH₂ 4 CH₂CO benzyl 16 CH₂CO₂H 20 6d(CH₂)₆NHCO₂[CH₂CH₂O]₇₀CH₂CH₂ 7 CH₂CO benzyl 16 CH₂CO₂H 17 6e 4 C═Obenzyl 16 H — 6f (CH₂)₆ 3 CH₂CO benzyl 10 CH₂CO₂H 27 6g(CH₂)₆NH[CO(CH₂)₅NH]₄— 2 C═O — — H — 6h (CH₂)₆NH[CO(CH₂)₅NH]₄— 1 C═ONH(CH₂)₆S-2-sialic acid  5 H — 6i — 0 C═O benzyl 16 H — 6j — 0 CH₂CObenzyl 16 CH₂CO₂H 24

Further examples of compounds of the invention include those in whichthe macromolecule M is a dextran or polyacrylic acid backbone, andwherein the substituents on the sialic acid group (2) are R2=Ac,R=guanidine and W=OCONH, and wherein the extra substituent Z is forexample a benzyl group, a biotin molecule or a fluorescein-containinggroup.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as to the treatment ofestablished infections or symptoms. Treatment is preferably commencedbefore or at the time of infection and continued until virus is nolonger present in the respiratory tract. In general a suitable dose of acompound of the invention will be in the range of 0.1 to 100 mg/Kg/day,preferably in the range of 0.2 to 20 mg/Kg/day.

Suitably treatment is given 1-4 times daily and continued for 3-7 dayspost infection. The desired dose may be given in a single dose, or asdivided doses at appropriate intervals.

While it is possible that, for use as a therapy, a compound of theinvention may be given as the raw chemical, it is generally preferableto present the active ingredient as a pharmaceutical formulation. Theinvention thus provides a pharmaceutical formulation comprisingcompounds of formula (II) or an acceptable salt or derivative thereof,together with one or more pharmaceutically acceptable carriers thereforand, optionally, other therapeutic and/or prophylactic agents.Pharmaceutical formulations include those for oral, nasal or topicaladministration or in a form suitable for inhalation or insufflation intothe respiratory tract. The formulations may, where appropriate, beconveniently presented in discrete dosage units and may be prepared byany of the methods well known in the art of pharmacy.

In general the compounds of the invention may be administered in theform of a solution or a suspension or as a dry powder. Foradministration to the respiratory tract according to the method ofinvention the neuraminidase inhibitors may be administered by any of themethods and formulations employed in the art for administration to therespiratory tract.

Solutions and suspensions will generally be aqueous and for exampleprepared from water alone or water and a physiologically acceptableco-solvent (for example ethanol, propylene glycol, polyethylene glycolssuch as peg 400). Such solutions or suspensions may additionally containother excipients for example preservatives (such as benzalkoniumchloride), solubilising agents/surfactants such as polysorbates (e.g.Tween 80), buffering agents, isotonically-adjusting agents (for examplesodium chloride), absorption enhancers and viscosity enhancers.Suspensions may additionally contain suspending agents (for examplemicrocrystalline cellulose, carboxymethyl cellulose sodium).

Solutions or suspensions are applied to the nasal cavity by conventionalmeans, for example with a dropper, pipette or spray. The formulationsmay be provided in single or multiple dose form. A spray may be achievedfor example by means of a metering atomising spray pump. Administrationto the respiratory tract may also be achieved by means of an aerosolformulation in which the compound is provided in a pressurised pack witha suitable propellant such as a chlorofluorcarbon (CFC) or othersuitable gas.

Alternatively the compounds may be provided in the form of a dry powder,for example a powder mix of the compound in a suitable base such aslactose, starch, starch derivatives and polyvinylpyrrolidine.Consequently the powder will form a gel in the nasal cavity. Informulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size, for example of the order of 5 microns or less. Sucha particle size may be obtained by means known in the art.

The compounds of the invention are prepared in several stages, the firstpart generally being the synthesis of a neuraminidase-binding sialicacid derivative of formula X—Y, wherein X and Y are as defined above.

Methods for the synthesis of sialic acid derivatives (2) with suitablefunctionality at the 7-position are described in British PatentApplication No. 9516276.4, and in International Patent Application No.PCT/AU97/00190.

Examples of suitable sialic acid derivatives X—Y are shown below inTable 5, wherein the groups R2, R and W are the substituents on moiety Xas described above.

TABLE 5 Compound No. R² R W Spacer Y 2a Ac Guanidine OCONH (CH₂)₆NH₂ 2bAc Guanidine OCONH (CH₂)₆NHCO(CH₂)₅NH₂ 2c Ac Guanidine OCONH(CH₂)₆NH(CO(CH₂)₅NH)₃CO(CH₂)₅NH₂ 2d Ac Guanidine OCONH(CH₂CH₂O)₂CH₂CH₂NH₂ 2e Ac Guanidine OCONH(CH₂)₆NH(CO(CH₂)₅NH)₃CO(CH₂)₅NH-Biotin 2f Ac Guanidine OCONH(CH₂)₆NHCONH(CH₂)₆NH₂ 2g Ac Guanidine OCONH(CH₂)₆NHCO₂[CH₂CH₂O]₇₀CH₂CH₂NH₂

The second part of the preparation of the compounds of the inventioninvolves attachment of the neuraminidase-binding units X—Y to themacromolecule M. For covalent attachment of units X—Y to macromoleculesM of a protein type, the conjugation can generally be carried out usingstandard cross-coupling methods which are well known (e.g. S. S. Wong,“Chemistry of Protein Conjugation and Cross-Linking” CRC Press, 1991; G.T. Hermanson, “Bioconjugate Techniques” Academic Press, 1996).

In the case of synthetic polymers, the units X—Y may be added to apreformed polymer backbone which has suitable activated substituents.For example, if group Y has a terminal amino functionality it may bereacted with activated ester substituents on a polyacrylate backbone.Alternatively, units X—Y with a suitable polymerisable substituent, suchas a terminal olefin may be polymerised or preferably co-polymerisedwith another olefin, to generate the macromolecular backbone. See forexample R. Roy, Trends in Glycoscience and Glycotechnology, 1996 879-99; N. V. Bovin and H. J. Gabius, Chem. Soc. Reviews., 1995 413)

EXAMPLE 1 Preparation of a GG167-Bovine Serum Albumin-Biotin conjugate(3a)

(a) Preparation of5-acetamido-7-(6′-(6″-aminocaproyl)aminohexyl)-carbamoyloxy-4-guanidino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonicAcid (2b) (7-(6-Aminocaproyl)amino-hexylcarbamoyloxy-GG167)

6-(t-Butyloxycarbonylamino)caproic acid (34 mg, 147 μmol) was dissolvedin a mixture of acetone (2.5 ml), water (100 μl), N-methyl morpholine (4μl, 36 μmol) and triethyl amine (21 μl, 147 μmol)). The solution wascooled to −12° C., and then isobutyl chloroformate (21 μl, 161 μmol) wasadded. The solution was stirred for 12 minutes.

Methyl5-acetamido-7-(6′-aminohexyl)-carbamoyloxy-4-guanidino-8,9-monocarbonyldioxy-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonatetrifluoroacetic acid salt (79 mg, 92 μmol) was dissolved inwater/acetone (1:1, 2 ml) containing triethylamine (40 μl, 287 μmol).This basic solution was cooled to 0° C. and added as a single portion tothe previous reaction mixture. The mixture was allowed to warm to roomtemperature and stirred for 4 hours.

The solvents were removed under reduced pressure. The crude product wastaken up in methanol/water (1:1, 4 ml) and then triethylamine (1 ml) wasadded. This solution was stirred overnight under argon. The solventswere removed on a rotary evaporator and the residue was dissolved inmethanol and adsorbed on to silica gel (1.5 g). Chromatography on silicagel (10 g), eluting with ethyl acetate/isopropanol/water (100/75/25)gave the product (53 mg, 77 μmol, 84%).

¹H n.m.r. (CD₃OD, 200 M Hz) δ5.67 (d, J 2.6 Hz, 1H); 5.03 (m, 1H); 4.62(m, 1H); 4.49 (m, 1H); 4.23 (m, 1H); 4.12 (m, 1H); 3.77 (m, 1H); 3.62(m, 1H); 3.12 (m, 6H); 2.22 (t, J 8 Hz, 2H); 2.0 (s, 3H); 1.5 (m, 23H).

The t-Boc protected sugar was dissolved in ethyl acetate/toluene andevaporated to dryness. The material was dissolved in trifluoroaceticacid (3 ml) and stirred at room temperature under argon for 1 hour. Thesolvent was removed under reduced pressure, and residual trifluoroaceticacid was removed by co-evaporation first with dichloromethane, then 3portions of water/methanol. The sample was then dissolved in water,filtered and lyophilised to give the product (141 mg, 152 μmol) as thetris TFA salt. Mass spectrum (FAB): 588 (M+1)

¹H n.m.r. (CD₃OD, 300 M Hz) δ5.92 (d, J 2.6 Hz, 1H) ; 5.01 (m, 1H); 4.60(dd, J 10, 3 Hz, 1H); 4.44 (dd, 9, 3 Hz, 1H); 4.23 (m, 1H); 4.05 (m,1H); 3.67 (dd, 13, 4 Hz, 1H); 3.52 (dd, 13, 7 Hz, 1H); 3.16 (m, 5H);2.96 (;t, 8 Hz, 2H); 2.25 (t, 8 Hz 2H); 2.00 (s, 3H); 1.7-1.3 (m, 14H).

(b) Preparation of the Conjugate between the GG167 Derivative (2b) andBovine Serum Albumin-(6-aminocaproyl biotin)₈

Protein coupling buffer: 0.1N NaHCO₃/0.2M NaCl

Dialysis buffer: 20 mM KH₂PO₄/0.15 M NaCl, pH 6.5

BSA-(caproyl-biotin)₈ (3 mg, 43 nmol, Pierce) was dissolved in thecoupling buffer (7.5 ml) and stirred gently for 30 minutes.

Bis-(N-hydroxy sulfosuccinimide)suberate (12 mg, 21 μmol, Pierce) wasdissolved in coupling buffer (1 ml) and added directly to a basicsolution of compound (2b) from Example 1(a) (19.5 mg, 21 μmol) incoupling buffer (1.5 ml). The reaction was allowed to stir for 7.5minutes. The BSA-biotin solution was then added dropwise to thederivatised-GG167 solution and the mixture was left to stir at roomtemperature for 1.5 hours. The solution was lyophilised, dissolved inwater (3.0 ml) and dialysed against dialysis buffer (3×1.5 L, cellulosetubing, 12,000 MW cut off).

The mixture was lyophilised, taken up in water (2.5 ml) and desalted ona PD-10 column (Pharmacia), then lyophilised to give the product (3a)(2.5 mg).

The estimation of the sugar incorporation (30 GG167 units per proteinmolecule) was based upon a colorimetric assay for the guanidine group(Sakaguchi Reaction, see Can. J. Chem., 1958 36 1541).

EXAMPLE 2 Preparation of Bovine Serum γ Globulin-(6-aminocaproylbiotin)₁₈-(GG167-7-carbamate-1,6-diaminohexane-6-aminocaproic AcidAmide-suberic Acid Amide)₆₀ (3b)

Bovine γ globulin (3 mg, 20 nmol, Sigma) was dissolved in couplingbuffer (1 ml) and stirred for 30 minutes.

N-Hydroxy sulfosuccinimidyl-N-Biotinyl-6-aminocaproate (1.8 mM, 280 μl,0.5 μmol) in coupling buffer was added to the protein solution, and thereaction was stirred at room temperature for 1 hour.

The reaction mixture was then diluted to 7.5 ml with coupling buffer andreacted with bis(N-hydroxy sulfosuccinimidyl)suberate andGG167-7-carbamate-1,6-diaminohexane-6-aminocaproic acid amide fromExample 1(a) above) under conditions identical to Example 1(b).

The yield of lyophilised γ-globulin conjugate (3b) was 2.5 mg.

EXAMPLE 3 Preparation of the Conjugate 3f between Compound 2c andHorseradish Peroxidase (HRP)

Compound 2c was coupled to HRP following the well established periodateoxidation methodology (see for example G. T. Hermanson, “BioconjugateTechniques” Academic Press, 1996 472) to give compound 3f.

HRP (type IV-A, Sigma Aldrich P-6782, 5 mg, 114 nmol) was dissolved insodium acetate/sodium chloride buffer (5 mM/150 mM, pH 4.5, 500 μl). Tothis was added freshly prepared sodium periodate solution (88 mM insodium acetate/sodium chloride buffer, 50 μl, 4.4 mmol) and the reactionwas allowed to stand in the dark at room temperature for 20 min. Themixture was chromatographed on a PD-10 column (Pharmacia Biotech,Sephadex G-25) pre-equilibrated with sodium acetate buffer (5 mM, pH4.5), and the eluent was freeze dried.

The oxidized HRP was dissolved in sodium carbonate buffer (0.2M, pH 9.5,1 ml) containing compound 2c (3.9 mg, 3.75 μmol) at 4° C., and thereaction was left to stand overnight at 4° C. A solution of sodiumcyanoborohydride (5M in 1N NaOH, 10 μl, 50 μmol) was added and thereaction allowed to stand overnight at 4° C. A solution of ethanolamine(1M, pH 9.5, 50 μl, 50 μmol) was added and the reaction allowed to standat room temperature for 30 min before chromatography on a PD-10 column,pre-equilibrated with distilled water. The eluent was freeze dried togive the HRP-compound 2c conjugate as a pale brown powder.

EXAMPLE 4 Preparation of Protein-GG167 Conjugates 3c, 3d and 3g-3j

Compounds 3c, 3d and 3g-3j were prepared by coupling the appropriateprotein with either compound 2c or 2g using bis(N-hydroxysulfosuccinimide)suberate and following a similar procedure to thatdescribed in Example 1, Part (b).

EXAMPLE 5 Preparation of a Biotinylated Complex (4d) between Avidin anda GG167-Biotin Conjugate

(a) Preparation of5-acetamido-7-(6′-(6″-(6′″-biotinylaminocaproyl)-triaminocaproyl)aminohexyl)-carbamoyloxy-4-guanidino-2,3,4,5-tetradeoxy-D-glycero-D-galacto-non-2-enopyranosonicacid (2e) was carried out using Boc-protected tetra(6-aminocaproic acid)and following a similar method to that described in Example 1.

(b) Avidin (3 mg, 0.0445 μmol) was dissolved in a solution of compound(2e) (0.5 mg, 0.434 μmol) in water (500 μl) at room temperature for 2hours. To this resulting solution were addedsulfo-N-hydroxysuccinimido-caproylamino-Biotin (Pierce #21335) (1000 μg,1.798 μmol) and a solution of sodium bicarbonate (1000 μg, 11.9 μmol) inwater (240 μl). The whole mixture was allowed to stand at roomtemperature for 45 minutes, then placed in a dialysis tube (molecularweight cut off 12,000). The tube was dialysed successively against 50 mMNaHCO₃ solution (4×250 ml) and water (8×250 ml), with the immersion timebeing 45 minutes in each case. The tube was finally dialysed againstwater (500 ml) at room temperature overnight. The resulting solutionfrom the dialysis tube was adjusted to pH 6.5-7.0 with sodiumbicarbonate and then freeze-dried to afford the title complex (4c) (3mg, 89%) as a white solid. The complex comprised four GG167-Biotinmolecules bound via the four avidin binding sites and then the avidinbackbone substituted with about eight to ten covalently-bound biotinligands. A diagram of the complex is shown in FIG. 1, wherein Arepresents avidin, B represents biotin and S represents the GG167 sugarmolecule.

EXAMPLE 6 Preparation of Polyacrylamides (5a)-(5e) Substituted withVarious GG167-ligands

(a) Poly(N-(acryloyloxy)succinimide) (pNAS) was prepared fromN-(acryloyloxy)succinimide as described by Ferruti et al. (Polymer,1972, 13, page 462). The ¹H n.m.r. spectrum and infrared spectrum of thepNAS were consistent with those previously reported in the literature. Asample of the batch of pNAS was converted into polyacrylamide bytreatment with conc. ammonia for several hours at room temperature andthe molecular weight of the dialysed polyacrylamide was found to beapproximately 50,000 using viscometry.

(b) Polyacrylamides (5a)-(5e) incorporating various GG167 derivativeswere prepared from the one batch of pNAS following the general methodoutlined below for compound (5c).

A solution of pNAS (10 mg, 59 μmol of NAS) in dimethylformamide (DMF,0.5 ml) was added, to a solution of compound (2f) (3.8 mg, 6.2 μmol) inDMF (0.5 ml) at room temperature with stirring. Triethylamine (10 μl, 70μmol) was added and the clear solution was stirred overnight at roomtemperature, then heated at 65° for five hours and stirred overnightagain at room temperature. Dilute aqueous ammonia (6 ml of 3% solution)was added to the reaction mixture and the clear solution was allowed tostand for 24 hours at room temperature. The reaction mixture wasevaporated to dryness under reduced pressure, and the residue wasdissolved in water (3 ml) and placed in dialysis tubing (MW cut-off12,000) and dialysed against water (500 ml, pH 6) for 24 hours. Thesolution was freeze-dried to give the GG167-containing polyacrylamide(5c) as a white solid (5 mg). The ¹H nmr spectrum (300 MHz) showed thefollowing broad signals: (D₂O) δ5.7, 4.4-4.6, 4.1, 3.3-3.7, 3.0,2.0-2.5, 1.9, 1.1-1.8. By comparison of the integral for the sialic acidprotons (δ5.7-3.2) with the integral for the polymer and spacer chains(δ1.0-3.2, minus the N-Acetyl peak at 1.9) the level of incorporation ofGG167 units was estimated to be about 10%.

EXAMPLE 7 Preparation of Polyacrylamide 51 which Has Both GG167 Ligandsand Biotin Ligands

A solution of pNAS (170 mg, 1 mmol of NAS) (MW about 50,000) in DMF (3ml) was stirred at room temperature, and a solution of compound 2a (TFAsalt, 30 mg, 50 μmol) and N-6-aminohexyl-biotinamide (7 mg, 20 μmol) inDMF (2 ml) was added. Triethylamine (50 μl) was added and the reactionmixture was stirred at 20° for 24 hours. Dilute ammonia (20 ml, 5%) wasadded to the reaction mixture and stirring continued for another 24hours. The reaction mixture was evaporated to dryness and the residuewas dissolved in 10 ml of water and dialysed in water for 2 days (1×4liters, tubing of 12,000 MW cut-off). With the Sakaguchi guanidinecolour test a positive result was obtained from the fluid remaining inthe tubing, but not from a concentrated sample of the last dialysiswater. The liquid from the dialysis tubing was freeze-dried to give 51as a cream fluffy solid (71 mg). The level of the ligends on the polymerwas estimated by NMR integration, and was consistent with almostcomplete incorporation of the starting amines.

EXAMPLE 8 Preparation of GG167-Containing Polyacrylamides 5i, 5k, 5m and5n

Compounds 5i, 5k, 5m and 5n were each prepared by reacting pNAS with theappropriate mixture of GG167 derivative (2a or 2c) and eitherbenzylamine, N-6-aminohexyl-fluorescein or aminoethanethiol, following asimilar procedure to that described in Example 7. For the preparation ofcompounds 5i and 5k (and also compounds 5h and 5j) pNAS of higherMW(>200 Kd) was used.

EXAMPLE 9 Preparation of Dextran (MW 500 KDa) with Multiple GG167(7-oxycarbamoylhexylamino-carbonyloxy-4-guanidino-Neu5Ac2en) (3.5 mole%) and N-benzylcarbamoyloxy (16.8 mole %) Substituents

To a solution of Dextran (MW 500,000) [100 mg, 0.617 mmol (based on aunit MW of 162)] in DMSO (5 ml) were added p-nitrophenyl chloroformate(510 mg, 2.53 mmol) and 4-dimethylaminopyridine (309 mg, 2.53 mmol). Themixture was stirred under argon, firstly at room temperature for 1 hr,then at 35˜40° C. for 3 hr. The resulting solution was combined with asolution of benzylamine (12.5 mg, 0.117 mmol) and4-dimethylaminopyridine (40 mg, 0.327 mmol) in pyridine (5 ml). Thereaction mixture was allowed to agitate under argon at room temperaturefor 16 hrs. before being evaporated under high vacuum to dryness. Theresidue was stirred in 2% potassium carbonate solution (25 ml) at 50° C.for 3 hrs. to produce a clear solution, which was then adjusted to pH7with 3M HCl. The resulting solution was dialyzed against water at roomtemperature for 3 days, freeze-dried to afford benzylated dextran (95mg, 83.4%) as a white solid containing 16.8 mole % ofoxycarbamoyl-methylenebenzene indicated by ¹H-nmr (D₂O)

To a solution of the benzylated dextran (5 mg, 0.027 mmol) in DMSO (0.25ml) were added p-nitrophenyl chloroformate (12.8 mg, 0.063 mmol) and4-dimethyl-aminopyridine (7.8 mg, 0.063mmol). The solution was stirredunder argon at room temperature for 1 hr, then at 35˜40° C. for 3 hrs.Afterwards it was combined with a solution of compound 2a (0.5 mg,0.00105 mmol) in a mixture of pyridine (0.25 ml) and DMSO (0.25 ml). Thereaction mixture was stirred at room temperature for 16 hr beforeevaporation under high vacuum to dryness. The residue was stirredvigorously in 1% potassium carbonate solution (5 ml) for 4.5 hr toproduce a clear solution. It was then adjusted to pH7.5 with 3M HCl,dialysed against water at room temperature for 24 hr and finallylyophilized to afford the title polymer (4.5 mg, 82%) as a white solid.¹H-nmr (D₂O) indicated that the polymer bore 16.8 molecules ofbenzylamine and 3.5 molecules of compound 2a per 100 glucose units ofthe carrier. The MW of the polymer was estimated as 623 KDa, and theaverage MW for one unit of7-aminohexylaminocarbonyloxy-4-guanidino-Neu5Ac2en was 5770.

EXAMPLE 10 Preparation of Multivalent GG167(7-oxyacetamidohexylaminocarbonyloxy-4-guanidino-Neu5Ac2en) (3 mole %),N-benzylacetamido-2-oxy (17 mole%) and 2-oxyacetate (20%) onDextran/5000 KDa

To a solution of Dextran (MW 500,000) [50 mg, 0.308 mmol (based on aunit MW of 162)] in water (0.3 ml) in an ice-bath was added potassiumhydroxide (138 mg, 2.46 mmol) in water (0.1 ml). The mixture was stirredat 0˜5° C. for 20 min. and then chloroacetic acid (102 mg, 1.07 mmol)was added. The resulting mixture was stirred at 70˜80° C. for 20 min.and then at room temperature for 2 hr. The reaction mixture was dilutedwith methanol (25 ml), the white precipitate collected by filtration,washed thoroughly with fresh methanol (25 ml) and dried. The wholeprocedure was repeated once to afford product as a white solidcontaining about 40 mole % of oxyacetate potassium salt as determined bytitration (52 mg, 84%).

To a solution of the acetic acid polymer (10 mg, 0.05 mmol) in water(0.4 ml) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (16 mg, 0.08 mmol) and sulfo-N-hydroxysuccinimide (17.4mg, 0.08 mmol). The mixture was stirred at room temperature for 20 min.and then combined with a solution of benzylamine (12.8 mg, 0.118mmol),in methanol (0.2 ml). The resulting mixture was stirred at roomtemperature for 3 hr and concentrated under vacuum to dryness. Theresidue was stirred in water (10 ml) containing NaHCO₃ (50 mg) at 50° C.to produce a clear solution, which was dialyzed against water for 3days, lyophilized to afford product (9 mg, 86%) as a white solidcontaining 17 mole % oxyacetamidomethlenebenzene as indicated by ¹H-nmr.

To a solution of the polymer (5 mg, 0.0239 mmol) in water (0.2 ml) wereadded 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (8mg, 0.04 mmol) and sulfo-N-hydroxysuccinimide (8.7 mg, 0.04 mmol). Themixture was stirred at room temperature for 20 min., then combined witha solution of compound 2a (1 mg, 0.0021 mmol) and4-dimethylaminopyridine (3 mg, 0.024 mmol) in pyridine (0.1 ml). Thereaction mixture was agitated at room temperature for 3 hr andevaporated to dryness. The residue was stirred in 1% NaHCO₃ solution (5ml) to produce a clear solution. After dialysis in water for 24 hr, thesolution was lyophilized to afford the title polymer as a white solid(4.5 g, 84%).

The acid titration and ¹H-nmr (D₂O) indicated that the polymer bore 17mole % of benzylamine, 3 mole % of GG167(7-aminohexylaminocarbonyloxy-4-guanidino-Neu5Ac2en) and 20 mole % ofacetate per 100 glucose units of the carrier. The MW of the polymer wasestimated as 683 KDa and the average MW for one polymeric7-amino-hexylamino-carbonyloxy-4-guanidino-Neu5Ac2en was 7420.

EXAMPLE 11 Preparation of polymeric-multivalent7-{6′-{6″-[6′″-(6″″-(6′″″-aminocaproyl)-aminocaproyl)-aminocaproyl]-aminocaproyl}-aminohexyl}-carbamoyloxy-4-guanidino-Neu5Ac2en(14 mole %) on Oxidized Dextran/500 KDa

To a solution of Dextran (MW 500,000) [20 mg, 0.123 mmol (based on aunit MW of 162)] in water (0.4 ml) at ice-bath temperature was addeddropwise sodium periodate (15.6 mg, 0.073 mmol) in water (0.4 ml). Thereaction mixture was stirred at 5° C. for 2 hr, then allowed to stir atroom temperature for 2 hr. The resulting mixture was diluted with water(1.2 ml) and passed through a Sephadex G-25 (10 ml) column. The columnwas eluted with water (3 ml). The eluate was freeze-dried to affordpartially oxidized Dextran (18 mg, 90%) as a white solid.

To a solution of the above oxidised Dextran (3 mg, 0.018 mmol) in water(0.6 ml) were added sodium bicarbonate (15 mg, 0.178 mmol) and compound2c.TFA salt (6 mg, 0.0057 mmol). The whole mixture was stirred at roomtemperature for 1 hr, then stirred in an ice-bath temperature. To thiscold mixture was added sodium cyanoborohydride (100 mg, 1.59 mmol) inportions. The reaction mixture was stirred at ice-bath temperature for 1hr, left at 5° C. for 16 hr, then was treated with more sodiumcyanoborohydride (100 mg, 1.59 mmol) and stirred at 5° C. for 1 hr, thenat room temperature for 2 hr, and finally dialysed against water for 24hr and lyophilized to give the title polymer (3 mg) as a white solid.

¹H-nmr (D₂O) indicated that the polymer bore about 430 molecules of7-{6′-{6″-[6″′-(6″″-(6′″″-aminocaproyl)-aminocaproyl)-aminocaproyl]-aminocaproyl}-aminohexyl-carbamoyl-oxy-4-guanidino-Neu5Ac2en(compound 2c) per partially oxidized Dextran molecule. Therefore the MWof the polymer was estimated to be 860 KDa and the average MW for eachunit of 4-guanidino-Neu5Ac2en was 2,000.

EXAMPLE 12 Preparation of Polymeric-multivalent7-suberamoyl-hexyl-carbamoyloxy-4-guanidino-Neu5Ac2en (5 mole %) onPolylysine/70˜150 KDa

A solution of 7-aminohexyl-carbamoyloxy-4-guanidino-Neu5Ac2en (compound2a, 6.4 mg, 0.0135 mmol) and disuccinimidyl suberate (5 mg, 0.0135 mmol)in a mixture of pyridine (0.1 ml) and DMF (0.1 ml) was stirred at 30° C.for 3 hr, then evaporated to dryness under high vacuum. The residue wastaken up in ether (10 ml×3) and dried to afford the activated ester.This was then combined with a solution of polylysine HBr salt(MW70,000-150,000) (10 mg, 0.0485 mmol) in a mixture of water (0.4 ml),DMSO (0.25 ml), DMF (0.6 ml), and pyridine (0.4 ml). The resultingmixture was stirred at room temperature for 10 hr, then dialyzed againstwater for 72 hr. The dialysate was diluted with water (10 ml), heated to50° C., before filtration. The filtrate was then lyophilized to affordthe title polymer (5 mg) as a white solid.

¹H-nmr (D₂O) indicated that the polymer bore 5 molecules of7-suberamoyl-hexylcarbamoyloxy-4-guanidino-Neu5Ac2en per 100 lysineunits of the carrier. Therefore the average MW for one polymeric4-guanidcino-Neu5Ac2en was 5140.

EXAMPLE 13 Preparation of Polymeric-multivalent7-{2′-[2″-(2″′-aminoethoxy)-ethoxy]-ethyl}-carbamoyloxy-4-guanidino-Neu5Ac2en(3.2 mole %) on Polyglutamic Acid/50˜100 KDa

To a solution of poly-glutamic acid sodium salt (MW50,000˜100,000) (10mg, 0.0657nnol) in water (0.6 ml) were added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.6 mg,0.050 mmol) and N-hydroxysulfo-succinimide (10.9 mg, 0.050 mmol). Themixture was stirred at room temperature for 25 min., then combined witha solution of7-{2′-[2″-(2′″-aminoethoxy)-ethoxy]-ethyl}-carbamoyloxy-4-guanidino-Neu5Ac2en.TFAsalt (compound 2d, 4.1 mg, 0.0081 mmol) in water (0.1 ml) and pyridine(0.1 ml). The resulting mixture was stirred at room temperature for 2hr. then dialyzed against water for 3 days, and finally lyophilized toafford the title polymer (9.5 mg) as a white solid.

¹H-nmr (D₂O) indicated that the polymer bore 3.2 molecules of7-{2′-[2″-(2″′-aminoethoxy)-ethoxy]-ethyl}-carbamoyloxy-4-guanidino-Neu5Ac2enper 100 glutamic acid units of the carrier. The average MW for onepolymeric 4-guanidino-Neu5Ac2en was 5250.

EXAMPLE 14 Preparation of N-hydroxyethylpolyacrylamide Conjugated withCompound 2c and Horseradish Peroxidase

Compound (2c)TFA salt (6 mg, 0.0057 mmol) was added to a solution ofpNAS (see Example 6, part b) (20 mg, 0.118 mmol of NAS) in DMF (1 ml).Triethylamine (20 μl) was added to the clear solution which was stirredat room temperature for 3 days. Half of the above reaction mixture wasadded to a solution of horseradish peroxidase (17 mg) in water (4 ml)and the mixture was left at 4° C. for several days. Ethanolamine (0.5 mlof a 10% solution in water) was added to the mixture to quench theremaining polyacrylate activated ester. After 2 hr the reaction mixturewas dialysed against water for 3 days and then the dialysate wasfreeze-dried to give the polymeric conjugate as a fluffy pale-brownpowder.

EXAMPLE 15 Determination of the Binding of the Compounds of theInvention to Influenza Virus Neuraminidase

Two influenza A viruses and one influenza B virus were used to test theability of the compounds to bind to whole virus influenza neuraminidase.The influenza A reassortants were eitherA/NWS/34-tern/Australia/G70C/75(H1N9) or A/NWS/Tokyo/3/67/H1N2 and theinfluenza B strain was B/Victoria/02/87. The neuraminidase assay wascarried out following a literature procedure (Potier, M., et al, Anal.Biochem., 1979 94 287), and the measured inhibition constants (IC₅₀) aresummarised in Table 6.

The inhibition constants for the macromolecules were calculated based onthe molecular weight per unit of attached GG167 derivative, e.g. thepolyacrylamide 5c which has a GG167-derived molecule bound to 10% of theacrylamide units was assigned a molecular weight of 1293.

TABLE 6 Binding Constants Against Influenza Virus Neuraminidase forCompounds of the Invention Compound No. NWS/Tokyo NWS/G70C B/Vic/02/873a 1 × 10⁻⁸ 7 × 10⁻⁹ 2 × 10⁻⁸ 3b 2 × 10⁻⁹ 2 × 10⁻⁹ — 3d 1 × 10⁻⁸ 1 ×10⁻⁸ — 4a 1 × 10⁻⁸ 9 × 10⁻⁹ 2 × 10⁻⁸ 4b 5 × 10⁻⁸ 3 × 10⁻⁸ 1 × 10⁻⁷ 4c 1× 10⁻⁷ 7 × 10⁻⁸ — 4d 1 × 10⁻⁷ 1 × 10⁻⁷ — 5a 3 × 10⁻⁸ 3 × 10⁻⁸ — 5b 1 ×10⁻⁸ 2 × 10⁻⁸ — 5c 1 × 10⁻⁸ 2 × 10⁻⁸ — 5d 4 × 10⁻⁸ 4 × 10⁻⁸ — 5e 2 ×10⁻⁸ 3 × 10⁻⁸ — GG167 (I) 1 × 10⁻⁹ 1 × 10⁻⁹ 1 × 10⁻⁸ DANA 6 × 10⁻⁶ 6 ×10⁻⁶ —

EXAMPLE 16 Detection of Influenza Virus on ELISA Plates Using CompoundNo. 4d

A virus solution (50 μl) of approximately 1×10⁸ pfu/ml of NWS/G70Cinfluenza A virus in phosphate-buffered saline (PBS) was added directlyinto the wells of a 96-well ELISA (Dynatech) plate, and the virus wasallowed to bind by standing overnight at 4° C. After washing, the plateswere blocked with PBS-Tween 20 according to standard procedures, andthen serial dilutions of Compound No. 4d were added to one of the ELISAplate rows, starting at 1 μM concentration of GG167 units and going downto 10⁻¹⁰ M. As a control, serial dilutions of a biotinylated monoclonalanti-neuraminidase NC10 antibody (L. C. Gruen, J. Immunological Methods,1994 168 91) were added to another row of the ELISA plate. Afterincubation for 1 hour the plates were washed to remove unbound compound,and then the virus was detected with Streptavidin-HRPO(Boehringer-Mannheim), using ABTS (Sigma) as the chromogenic substrateand about thirty minutes incubation.

Concentrations of Compound No. 4d above 10⁻⁹ M allowed detection of thevirus, and an increasingly strong signal was observed, in parallel withincreasing compound concentration. Thus the approximately 5×10⁶ virusparticles per well could readily be detected with Compound No. 4d. Thebiotinylated antibody control gave a similar level of signal.

EXAMPLE 17 Capture and Detection of a Complex between Influenza Virusand Compound No. 3a

To allow coating of virus particles with the GG167-biotin derivative, 10μl of a solution of the NWS/G70C influenza A virus (1×10⁸ pfu/ml) waspre-incubated for 1 hour with various concentrations of compound 3a inthe wells of separate rows of an ELISA plate. Half log₁₀ dilutions ofcompound No. 3a were used, starting from 1 μM concentration of GG167units and going down to 0.00001 μM. The virus-compound complexes werethen transferred to an ELISA plate which had been pre-coated with avidinand incubated for 1 hour to allow capture. The plates were washed inPBS-Tween 20, and the captured virus was detected with a polyclonalrabbit antibody directed to the virus hefagglutinin according tostandard procedures.

The best result was found with a 0.1 μM concentration of Compound No.3a, which clearly allowed detection of virus at 10⁶ pfu. At higherconcentrations of compound the signal was weaker, possibly due to theblocking of some avidin sites by free compound No. 3a, whilst at lowerconcentrations (<0.001 μM) the detection signal was also weaker,probably due to there being insufficient compound to bind fully to allof the virus particles.

EXAMPLE 18 Direct Detection of Influenza Virus-GG167-protein-biotinConjugate with Streptavidin-Horseradish Peroxidase

Following the same procedure as described in Example 17 above and usingcompound 4d, the direct detection of bound virus withstreptavidin-horseradish peroxidase, instead of the anti-hemagglutininantibody, was also observed.

EXAMPLE 19 Inhibition of Influenza Hemagglutination by Macromolecules ofthe Invention

Compounds of the invention were tested for the ability to inhibithemagglutination (HAI) of influenza strains X-31 (H3N2), G70C and TokyoA, following the standard type of method that has been described in theliterature (see for example J. Amer. Chem. Soc., 1997 119 4103 andreferences cited therein). The HAI assay was performed using solutionsof the polymeric GG167 conjugates in PBS which were 2-fold seriallydiluted across 12 microtitre plate wells. Suspension of virus, dilutedto 4 HA units in PBS were added to the wells. After 2 hours at 4° C.0.5% suspension of chicken erythrocytes was added to each well. After 1hours at 4° C., the lowest concentration of inhibitor that preventedagglutination of the erythrocytes was determined. The results aresummarised in Table 7.

TABLE 7 Lowest Concentration (μM of GG167 units) that InhibitedAgglutination Compound No. G70C TOKYO X-31 Polyacrylamide 187.5 187.593.75 (assumed Mw 20,000 unsubstituted) 5f 7.8 7.8 7.8 5g 11.7 31.2523.4 6c 2.93 — 3. 91 Fetuin (control) 0.73 5.86 <0.48

EXAMPLE 20 Inhibition of Influenza Virus Replication by Macromoleculesof the Invention

Compounds of the invention were tested for the ability to inhibit thereplication of influenza A virus following the standard method that hasbeen described in the literature (see for example Watanabe et al, J.Virological Methods, 1994, 48, 257). The assay was carried out usingMDCK cells, and the results are shown in Table 8 below. The results areshown as the minimum compound concentration that inhibits cytopathiceffect by 50% [ID₅₀ (μg/ml)], calculated by using a regression analysisprogram for semilog curve fitting. The results show that all of themacromolecules which have GG167 derivatives attached to them are moreactive against influenza virus than the unsubstituted backbones bythemselves. The results also show that many of the polymeric compoundsare more active than the simple monomeric ligand (compound 2c),particularly when calculated on the basis of the molar concentration ofGG167 units. The Therapeutic Index for each compound was calculated bydividing the minimum cytotoxic drug concentration (MTC) by the ID₅₀.

TABLE 8 Compound (Molecular weight per GG167 unit) ID₅₀ (μg/ml) MTCTherapeutic Index GG167 (332) <0.32 >100 >312.5 2c (1,040)3.0 >100 >32.8 3d (10,000) 1.24 >100 >80.9 5a (1,451) <0.32 >100 >312.55g (2,400) 1.48 >100 >67.4 5h (4,500) 1.22 >100 >81.8 5i (5,000)1.11 >100 >89.6 5j (4,000) 3.20 >100 >31.2 5k (4,500) 0.46 >100 >215.6Polyacrylamide 94.7 >100 >1.06 (unsubstituted) 6a (5,770)26.7 >100 >3.75 6c (5,700) 0.31 >100 >322.3 6d (6,800) 3.00 >100 33.3 6e(8,480) 21.9 >100 4.56 6f (7,270) 7.2 >100 >13.9 6g (11,750)43.6 >100 >2.29 6h (17,850) 23.6 >100 >4.24 6i (unsubst. >100 >100 —benzyldextran) 6j (unsubst. >100 >100 — benzyldextran) Example 11(1,980) <0.32 >100 >312.5 Example 12 (5,140) 0.45 100 221.1 Example 13(5,250) 0.39 >100 >258.7 Ribavirin 1.0-2.6 >32 >12.22-31.98

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purpose of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

Reference cited herein are listed on the following pages, and areincorporated herein by this reference.

What is claimed is:
 1. A macromolecular compound comprising amacromolecule attached to at least one sialic acid derivative via theC-7 position of the sialic acid derivative structure, wherein the sialicacid derivative binds to the active site of influenza virusneuraminidase but is not cleaved by the neuraminidase.
 2. Amacromolecular, compound according to claim 1, in which the sialic acidderivative is attached to the macromolecule via a spacer or linkergroup.
 3. The macromolecular compound of claim 2, in which the spacer orlinker group Y is selected from the group consisting of aminoalkylgroups, (poly)amino acids, linear peptides, oligosaccharides andpolysaccharides, polyethylene glycol units, and aminodialkylureas, orany combination of these groups.
 4. A macromolecular compound accordingto claim 1, in which the sialic acid derivative inhibits influenza virusneuraminidase with an inhibition constant (IC₅₀) of 10⁻⁶ M or less.
 5. Amacromolecular compound according to claim 1, in which the sialic acidderivative is compound (I)

functionalised via the 7-position of the sialic acid structure.
 6. Apharmaceutical composition comprising a compound according to claim 5,together with a pharmaceutically-acceptable carrier, in which thecompound has an ID₅₀ for neuraminidase of less than 5 μg/ml.
 7. Acomposition according to claim 6, further comprising one or moreadditional therapeutically active agents.
 8. A composition according toclaim 7, in which the additional therapeutically active agent is ananti-viral agent.
 9. A method of treating influenza infection in amammal, the method comprising administering to the mammal thepharmaceutical composition of claim
 6. 10. The method of claim 9,wherein the mammal is a human.
 11. The macromolecular compound of claim5, wherein the sialic acid derivative is attached to the macromoleculevia a spacer or linker group.
 12. The macromolecular compound of claim11, in which the spacer or linker group is selected from the groupconsisting of an aminoalkyl group, a (poly)amino acid, a linear peptide,an oligosaccharide, polyethylene glycol, and aminodialkylurea, or anycombination of these groups.
 13. A method of treating influenzainfection in a mammal, the method comprising administering to the mammalan effective amount of the compound of claim
 5. 14. The method of claim13, wherein the mammal is a human.
 15. A macromolecular compoundaccording to claim 1 of formula (II): (X—Y)_(n)—M—(Z)_(m)  (II) whereinX is a neuraminidase-binding 2,3-dehydro-sialic acid derivative (2);

which is linked at the W via a spacer group Y to a macromolecule M, andZ is an optional extra substituent on the macromolecule; wherein Rrepresents an azido group, an unsubstituted or substituted guanidinogroup, or an unsubstituted or substituted amino group; R² representsCOCH₃, COCF₃, SO₂CH₃ or SO₂CF₃; W represents O(C═O)NH, O(C═S)NH,NH(C═O)NH or NH(C═S)NH and is attached through the NH to the spacergroup Y; m is an integer between 0 and 1000; in is an integer between 1and 1,000; the spacer group Y is an optionally substituted chain of upto 1000 atoms chosen from carbon, nitrogen, oxygen and sulphur; and themacromolecule M is a synthetic or natural polymer, protein, antibody orenzyme of molecular weight from 10⁴ up to 10⁷, linked covalently ornon-covalently to the spacer group Y.
 16. A macromolecular compoundaccording to claim 15, in which Z is present and is a group which bindshemagglutinin.
 17. A macromolecular compound according to claim 15, inwhich the spacer group Y is selected from the group consisting ofaminoalkyl groups, (poly)amino acids, linear peptides, oligosaccharidesand polysaccharides, polyethylene glycol units, and aminodialkylureas,or any combination of these groups.
 18. A macromolecular compoundaccording to claim 17, in which the spacer group Y has a terminalamino-group.
 19. A macromolecular compound according to claim 17, inwhich all spacer groups Y in the compound are identical.
 20. Amacromolecular compound according to claim 17, in which the spacergroups Y are a combination of moieties selected from the groupconsisting of aminoalkyl groups, (poly)amino acids, linear peptides,oligosaccharides, polysaccharides, polyethylene glycol units, andaminodialkylureas.
 21. A macromolecular compound according to claim 15,in which the macromolecule M is selected from the group consisting ofproteins, enzymes, antibodies, water-soluble synthetic polymers,polysaccharides and polyaminoacids.
 22. A macromolecular compoundaccording to claim 15, in which the macromolecule M is selected from thegroup consisting of bovine serum albumin, horseradish peroxidase (HRP),avidin, streptavidin, neutravidin, and immunoglobulins.
 23. Amacromolecular compound according to claim 15, in which themacromolecule M is selected from the group consisting ofpolysaccharides, polyacrylamides, polyethylene glycols, polyureas,polyacids, polyesters, polyamides and N-(2 hydroxypropyl)methacrylamide(HMPA), said macromolecule being pharmaceutically acceptable.
 24. Amacromolecular compound according to claim 15, in which R is a guanidinoor amino group substituted with a methyl, ethyl, allyl, amino, cyano ornitro group.
 25. A macromolecular compound according to claim 15, inwhich X is a compound of formula (2), in which R is guanidine, R² isacetyl, W is the group O(═CO)NH, and the spacer group Y is a chain madeup of between 6 and 60 carbon, nitrogen and oxygen atoms.
 26. Apharmaceutical composition comprising a compound according to claim 15together with a pharmaceutically-acceptable carrier, in which thecompound has an ID₅₀ for influenza virus neuraminidase of less than 5μg/ml.
 27. A composition according to claim 26, further comprising oneor more additional therapeutically active agents.
 28. A compositionaccording to claim 27, in which the additional therapeutically activeagent is an anti-viral agent.
 29. A method of treating influenzainfection in a mammal, the method comprising administering to the mammalthe pharmaceutical composition of claim
 26. 30. The method of claim 29,wherein the mammal is a human.
 31. A method of treating influenzainfection in a mammal, the method comprising administering to the mammalan effective amount of the compound of claim
 15. 32. The method of claim31, wherein the mammal is a human.
 33. A pharmaceutical compositioncomprising a compound according to claim 1, together with apharmaceutically-acceptable carrier, in which the compound has an ID₅₀for neuraminidase of less than 5 μg/ml.
 34. A composition according toclaim 33, further comprising one or more additional therapeuticallyactive agents.
 35. A composition according to claim 34, in which theadditional therapeutically active agent is an anti-viral agent.
 36. Amethod of treating influenza infection in a mammal, the methodcomprising administering to the mammal the pharmaceutical composition ofclaim
 33. 37. The method of claim 36, wherein the mammal is a human. 38.A method of treating influenza infection in a mammal, the methodcomprising administering to the mammal an effective amount of a compoundaccording to claim
 1. 39. The macromolecular compound of claim 1, inwhich the macromolecule is selected from the group consisting of aprotein, an enzyme, an antibody, a water-soluble synthetic polymer, apolysaccharide and a polyaminoacid.
 40. The macromolecular compound ofclaim 1, in which the macromolecule is selected from the groupconsisting of bovine serum albumin, horseradish peroxidase (HRP),avidin, streptavidin, neutravidin, and an immunoglobulin.
 41. Themacromolecular compound of claim 1, in which the macromolecule is asselected from the group consisting of a polysaccharide, apolyacrylamide, a polyethylene glycol, a polyurea, a polyacid, apolyester, a polyamide and N-(2 hydroxypropyl)methacrylamide (HMPA).